Plant virus particles for delivery of antimitotic agents

ABSTRACT

Anti-lymphoma plant virus particles are described. The anti-lymphoma plant virus particles include a filamentous or rod-shaped plant virus particle linked to an antimitotic agent. A therapeutically effective amount of an anti-lymphoma plant virus particle can be administered to a subject to provide a method of treating lymphoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/193,186, filed on Jul. 16, 2015, the disclosure of which ishereby incorporated by reference in its entirety.

BACKGROUND

Lymphoma can be divided into two categories: Hodgkin's lymphoma, andNon-Hodgkin's lymphoma. Hodgkin's disease is a specific kind of lymphomacommonly diagnosed by the appearance of Reed-Sternberg cells in tissuebiopsies. Fortunately, Hodgkin's lymphoma is one of the most curableforms of cancer, and patients with this disease have a 5-year survivalrate of 85%. However, Non-Hodgkin's lymphoma is the more common type oflymphoma, and covers a range of lymphatic cancers, including: diffuselarge B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma,mantle cell lymphoma, marginal zone B-cell lymphoma, burkitt lymphoma,lymphoplasmacytic lymphoma, hairy cell leukemia, primary central nervoussystem lymphoma, precursor T-lymphoblastic lymphoma and peripheralT-cell lymphomas.

The most common form of non-Hodgkin's lymphoma is diffuse large B-celllymphoma. Patients with this disease have poor survival with a 5 yearsurvival rate as low as 58%. The treatment success rates and prognosisheavily depend on the stage at which the disease is diagnosed. Both,Hodgkin's and non-Hodgkin's lymphoma are staged according to the AnnArbor staging system, which stages a cancer from I to IV. The numberrepresents the extent to which the cancer has spread, with stages I-IIIrepresenting one to three cancerous lymph nodes, respectively. Stage IVmarks disseminated disease that has spread into secondary organs awayfrom the main sites of disease. Depending on the disease staging at thetime of diagnoses, the treatment regime differs: For example, forpatients with non-Hodgkin's lymphoma stage I/II, the clinical protocolcalls for a combination therapy of Rituximab, cyclophosphamide,vincristine, doxorubicin, and prednisone (R-CHOP) for 3-4 cycles. In theadvanced stages III and IV, R-CHOP is administered for 6 cycles,sometimes with involved-field radiation therapy (IFRT). In cases ofrelapse, the patient may be administered platinum based chemotherapy,radio-immunotherapy, and higher doses of previous chemotherapeutics.Chen et al., Expert Opin. Drug Deliv., vol. 2, no. 5, pp. 873-890(2005).

It is clear that novel and innovative therapeutic strategies are neededto increase the survival rates as well as to mitigate the adverse sideeffects associated with many of the treatment regimes described above.Key strategies include to device methods to lower the effective dose ofsystemically administered toxic drugs—this can be achieved throughtargeted drug delivery strategies thereby increasing the partitioning ofthe drug to the site of the disease.

One avenue of disease-specific drug targeting is found in theapplication of antibodies, and more specifically antibody-drugconjugates (ADCs). Bouchard et al., Bioorg. Med. Chem. Lett., vol. 24,no. 23, pp. 5357-5363 (2014). Antibodies can be selected to virtuallyany disease target; the antibody itself can be therapeutic or can carrya therapeutic cargo. Drug-targeting my means of antibody target-antigenspecificity holds great potential for cancer therapy. Tumor neoantigenshave been discovered allowing the selection of target-specificantibodies, therefore allowing drug targeting Immunotherapies aregaining momentum not only in clinical trials, but such biologicaltherapies have become a clinical reality. A notable example is themonoclonal antibodies therapy rituximab. Rituximab targets CD20, whichis a protein expressed on most non-Hodgkin's B-cell lymphoma. The CD20target is not normally found in circulation, therefore making thistherapy highly specific. Rituximab is the standard for patientsdiagnosed with non-Hodgkin's B-cell lymphoma, yet the survival rates areonly 58%, indicating there is still room for improvement. Feugier etal., J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol., vol. 23, no. 18, pp.4117-4126 (2005).

Another avenue toward targeted therapies is the development ofnanoparticles. Nanoparticles typically measure between 10-500 nm and arethus small enough to efficiently navigate circulation, traffic throughtissues and target and enter cells. Albanese et al., Annu. Rev. Biomed.Eng., vol.14, no. 1, pp. 1-16 (2012); Yildiz et al., Curr. Opin.Biotechnol., vol. 22, no. 6, pp. 901-908 (2011). Nanoparticles arelarger than antibodies and offer multivalency; i.e., while an IgGantibody offers two binding sites for antigen binding, a nanoparticlehas the potential to bind to multiple hundred-to-thousands of bindingsites. The multivalency provides a mechanism to increase targetspecificity through added avidity effects. Furthermore, multifunctionaldesigns are possible, where toxic payloads and/or contrast agents areloaded into the nanoparticle while targeting ligands enabletissue-specific delivery with increased payload delivery. M. Wu and Z.Niu, Nanoparticles for Biotherapeutic Delivery (Volume 1), FutureScience Ltd, pp. 50-62 (2015); Xiao et al., Cancer Res., vol. 72, no. 8,pp. 2100-2110, (2012).

Nanoparticles take advantage of their size and shape to gain increaseduptake into tumor vasculature. Rapid angiogenesis occurs to supply thetumor with nutrients and oxygen and support the increased growth—as aresult the neovasculature is leaky with a porous endothelium. This leakyvasculature with pores at the nano-to-micron size, create the perfectentry ways for nanoparticles to enter the tumor. Wong et al., PLoS ONE,vol. 10, no. 5, (2015) Simultaneously, the microenvironment created bythe angiogenesis causes local compressive forces, which in turn lead topoor lymphatic drainage. This effect is known as the enhancedpermeability and retention effect (EPR). Simply by flowing through thebloodstream, the nanoparticles are likely to extravasate into the tumor,and stay in that environment due to the EPR effect. Some nanoparticles,such as doxil, a liposomal formulation of doxorubicin, have beenclinically approved for treatments in ovarian cancer, AIDS-relatedKaposi sarcoma, and multiple myeloma. Nevertheless, while the researchdevelopment pipeline is moving rapidly, nanoparticle therapies have notyet made it into the standard of care for non-Hodgkin's-lymphoma. Rinket al., Curr. Opin. Oncol., vol. 25, no. 6, pp. 646-651 (2013). Ofcourse, the EPR effect does not hold count for blood cancers such asnon-Hodgkin's-lymphoma. Alternative methods must be developed to targetpotent therapies to this disease.

SUMMARY

Nanoparticle drug therapies have shown great promise enabling drugdelivery to sites of disease based on their size, shape and surfaceengineerability. Viral nanoparticles are biology-derived carriers thatare growing in popularity due to their simple genetic and chemicalmodification, size tunability from spherical to high-aspect ratio, andbiocompatibility. These attributes give a wide range of tools forengineers to design therapeutics with specific toxic loads and surfacechemistries to efficiently navigate the body. The inventors havedeveloped a viral nanoparticle using the nucleoprotein component fromthe tobacco mosaic virus (TMV) as the carrier for delivery of theantimitotic drug valine-citrulline monomethyl auristatin E (vcMMAE). Inaddition, they have demonstrated successful synthesis of theformulation, and the effective cell killing of Non-Hodgkin's lymphoma invitro.

Accordingly, in one aspect, the present invention provides ananti-lymphoma virus particle, comprising a filamentous or rod-shapedplant virus particle linked to an antimitotic agent. In someembodiments, the plant virus particle is a tobacco mosaic virus, whilein other embodiments the antimitotic agent monomethyl auristatin E(MMAE).

In another aspect, the present invention provides a method of treatinglymphoma in a subject by administering to the subject a therapeuticallyeffective amount of an anti-lymphoma virus particle, comprising afilamentous or rod-shaped plant virus particle linked to an antimitoticagent. In some embodiments, the lymphoma is non-Hodgkin's lymphoma,while in further embodiments the anti-lymphoma virus particle isadministered together with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing drawings.

FIG. 1A and FIG. 1B provide images showing the structure of tobaccomosaic virus (TMV). Chimera image shows the 300 nm by 18 nm rod with topview (1A) and side view (1B). Coat protein subunit in lower righthighlights the reactive groups, glutamines on the interior, and lysineon the exterior.

FIG. 2A and FIG. 2B provide schemes showing the chemical structures andbioconjugation reaction. A) Bioconjugation scheme steps: FirstTMV-lysine reacts with SATP via NHS chemistry, next TMV-SATP's thiolgroup is deprotected using hydroxylamine, followed by reaction of TMV-SHwith the maleimide group of vcMMAE, resulting in TMV-MMAE. B) thechemical structure of vcMMAE.

FIGS. 3A and FIG. 3B and FIG. 3C provide graphs and images showing thebiochemical characterization of the vcMMAE-loaded TMV. A) SDS-gel: lanesLane 1) TMV-lys, Lanes 2 and 4) TMV-SATP, Lanes 3 and 5) TMV-vcMMAE. B)Densinometric analysis showing two distinct bands; CP vs CP-vcMMAE. C)Transmission electron microscopy (TEM) of negatively-stained TMV-vcMMAE.

FIG. 4 provides images of Karpas 299 cell interactions with TMV. DAPI:DAPI stain showing the nuclei. Lamp1: Endolysosomes stained with mouseanti-human Lamp-1 antibody, and secondary Alexa Fluor 488 goatanti-mouse antibody. TMV: TMV particles stained with primary rabbitantibody alpha-TMV, and anti-rabbit-555. Overlay: All three labelssimultaneously shown, with the colocalization of TMV and theendolysosome.

FIG. 5 provides a graph showing the results of the cell viability assay.Cell viability assay using Karpas 299 cells after treatment with vcMMAE(circles) and TMV-vcMMAE (triangles). Cell viability was determinedafter 72 hours using Alamar Blue assay. Error bars were calculated bystandard deviation (experiments were done in triplicates). Data wasanalyzed and graphed using Prism® v6.0b software.

DETAILED DESCRIPTION

The present invention provides anti-lymphoma plant virus particles thatare a filamentous or rod-shaped plant virus particle linked to anantimitotic agent. In an additional aspect of the invention, atherapeutically effective amount of an anti-lymphoma plant virusparticle is administered to a subject to provide a method of treatinglymphoma.

Definitions

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. As used in this specificationand the appended claims, the singular forms “a”, “an” and “the” includeplural references unless the content clearly dictates otherwise. Thus,for example, reference to “a virus particle” includes a combination oftwo or more virus particles, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or 110%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a subject afflicted with a condition ordisease such as lymphoma, including improvement in the condition throughlessening or suppression of at least one symptom, delay in progressionof the disease, etc.

Prevention, as used herein, refers to any action providing a benefit toa subject at risk of being afflicted with a condition or disease such aslymphoma, including avoidance of the development of lymphoma or adecrease of one or more symptoms of the disease should lymphoma develop.The subject may be at risk due to exposure to a carcinogen, or as aresult of family history.

A “subject,” as used herein, can be any animal, and may also be referredto as the patient. Preferably the subject is a vertebrate animal, andmore preferably the subject is a mammal, such as a domesticated farmanimal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In someembodiments, the subject is a human.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects in light ofthe severity of the disease and necessity of the treatment.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of decreasing disease severity while avoiding adverse side effectssuch as those typically associated with alternative therapies. Thetherapeutically effective amount may be administered in one or moredoses.

“Targeting,” as used herein, refers to the ability of filamentous or rodshaped plant virus particles to be delivered to and preferentiallyaccumulate in cancer tissue in a subject.

In one aspect, the invention provides an anti-lymphoma virus particle,comprising a filamentous or rod-shaped plant virus particle linked to anantimitotic agent. Providing anti-lymphoma plant virus particle with ananti-mitotic agent linked to the plant virus particle helps protect theanti-mitotic agents from degrading or having toxic effects in thebloodstream, while allowing their release upon degradation of the virusparticles within lymphoma cells.

Filamentous and Rod-shaped Plant Viruses

A filamentous plant virus is a virus that primarily infects plants andhas a non-enveloped filamentous structure. A filamentous structure is along, thin virion that has a filament-like or rod-like shape that ismuch longer than it is wide and therefore has a high-aspect ratio. Forexample, Alphaflexiviridae have a length of about 470 to about 800 nm,and a diameter of about 12-13 nm. Filament-like virus particles areflexible in addition to being long and thin, and therefore someembodiments of the invention are directed to use of a flexiblefilamentous plant virus.

In some embodiments, the filamentous plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, thefilamentous plant virus belongs to the Alphaflexiviridae family. TheAlphaflexiviridae family includes the genus Allexivirus, Botrexvirus,Lolavirus, Mandarivirus, Potexvirus, and Sclerodamavirus. In someembodiments, the filamentous plant virus belongs to the genusPotexvirus. In further embodiments, the filamentous plant virus belongsto the Potato Virus X species.

A rod-shaped plant virus is a virus that primarily infects plants, isnon-enveloped, and is shaped as a rigid helical rod with a helicalsymmetry. Rod shaped viruses also include a central canal. Rod-shapedplant virus particles are distinguished from filamentous plant virusparticles as a result of being inflexible, shorter, and thicker indiameter. For example, Virgaviridae have a length of about 200 to about400 nm, and a diameter of about 15-25 nm. Virgaviridae have othercharacteristics, such as having a single-stranded RNA positive sensegenome with a 3′-tRNA like structure and no polyA tail, and coatproteins of 19-24 kilodaltons.

In some embodiments, the rod-shaped plant virus belongs to a specificvirus family, genus, or species. For example, in some embodiments, therod-shaped plant virus belongs to the Virgaviridae family TheVirgaviridae family includes the genus Furovirus, Hordevirus,Pecluvirus, Pomovirus, Tobamovirus, and Tobravirus. In some embodiments,the rod-shaped plant virus belongs to the genus Tobamovirus. In furtherembodiments, the rod-shaped plant virus belongs to the tobacco mosaicvirus species. The tobacco mosaic virus has a capsid made from 2130molecules of coat protein and one molecule of genomic single strand RNA6400 bases long. The coat protein self-assembles into the rod likehelical structure (16.3 proteins per helix turn) around the RNA whichforms a hairpin loop structure. The protein monomer consists of 158amino acids which are assembled into four main alpha-helices, which arejoined by a prominent loop proximal to the axis of the virion. Virionsare ˜300 nm in length and ˜18 nm in diameter. Negatively stainedelectron microphotographs show a distinct inner channel of ˜4 nm.

Filamentous and rod-shaped plant virus particles have an interior and anexterior. The exterior of a plant virus particle is the portion of thevirus particle that is directly exposed to the environment. The interiorof the plant virus particle is the portion of the virus particle thattypically is adjacent to the genomic material within the virus particle,and is not directly exposed to the environment. In some embodiments, theplant virus particles are genetically modified to have one or moreadditional attachment sites on the interior or exterior of the plantvirus particle. For example, the interior or exterior of the plant virusparticle can be modified to include one or more additional lysineresidues.

Antimitotic Compounds

Antimitotic compounds are drugs that inhibit mitosis, or cell division,and typically function by disrupt microtubules, which are structuresthat pull cells apart during cell division. Antimitotic compounds areuseful for treating lymphoma because cancer cells are able to grow andeventually spread through the body (metastasize) through continuousmitotic division, and are therefore more sensitive to inhibition ofmitosis than normal cells.

There are a wide variety of known antimitotic compounds which can belinked to plant virus particles according to the present invention.Classes of antimitotic compounds include taxanes, vinca alkaloids, anddolastatins. Examples of taxanes include paclitaxel and docetaxel, whileexamples of vinca alkaloids include vinblastine, vincristine, vindesine,and venorelbine. Dolastatins are compounds identified in the extracts ofthe sea hare Dolabella auricularia that have shown pronouncedantineoplastic and antimitotic activity. Examples of dolastatins includedolastatin 10, dolastatin 11, dolastatin 15, and monomethyl auristatin E(MMAE), which is a dolastatin 10 derivative. A further dolastatin isdolastatin is valine-citrulline monomethyl auristatin E (vcMMAE), whichis an analog of MMAE that has been modified to facilitate easyattachment to proteins such as antibodies. Other examples of antimitoticcompounds include colchicine, podophyllotoxin, and glaziovianin A.

Association of Antimitotic Agents with the Plant Virus Particles

The present invention provides an anti-lymphoma virus particle,comprising a filamentous or rod-shaped plant virus particle linked to anantimitotic agent. The antimitotic agent can be linked to either theinterior of the plant virus particle, the exterior of the plant virusparticle, or to both the interior and exterior of the plant virusparticle. An antimitotic agent is linked to the plant virus particle bybeing chemical bonded to the plant virus particle, though the linkagecan be either direct or indirect, where indirect linkage is through anintermediate linking molecule.

In general, antimitotic agents can be conjugated to the filamentous orrod-shaped plant virus particles by any suitable technique, withappropriate consideration of the need for pharmacokinetic stability andreduced overall toxicity to the patient. The term “conjugating” whenmade in reference to an antimitotic agent and a plant virus particle asused herein means covalently linking the agent to the virus subject tothe single limitation that the nature and size of the agent and the siteat which it is covalently linked to the virus particle do not interferewith the biodistribution of the modified virus.

An agent can be coupled to a filamentous or rod-shaped plant virusparticle either directly or indirectly (e.g. via a linker group). Insome embodiments, the agent is directly attached to a functional groupcapable of reacting with the agent. For example, viral coat proteinsinclude lysines that have a free amino group that can be capable ofreacting with a carbonyl-containing group, such as an anhydride or anacid halide, or with an alkyl group containing a good leaving group(e.g., a halide). Viral coat proteins also contain glutamic and asparticacids. The carboxylate groups of these amino acids also presentattractive targets for functionalization using carbodiimide activatedlinker molecules; cysteines can also be present which facilitatechemical coupling via thiol-selective chemistry (e.g.,maleimide-activated compounds). In addition, genetic modification can beapplied to introduce any desired functional residue, includingnon-natural amino acids, e.g. alkyne- or azide-functional groups. SeePokorski, J. K. and N. F. Steinmetz Mol Pharm 8(1): 29-43 (2011).

Alternatively, a suitable chemical linker group can be used. A linkergroup can serve to increase the chemical reactivity of a substituent oneither the agent or the virus particle, and thus increase the couplingefficiency. Preferred groups suitable for attaching agents to virusparticles are lysine residues present in the viral coat protein.Suitable linkage chemistries include maleimidyl linkers and alkyl halidelinkers and succinimidyl (e.g., N-hydroxysuccinimidyl (NHS)) linkers(which react with a primary amine on the filamentous or rod-shaped plantvirus particle). Several primary amine and sulfhydryl groups are presenton viral coat proteins, and additional groups can be designed intorecombinant viral coat proteins. It will be evident to those skilled inthe art that a variety of bifunctional or polyfunctional reagents, bothhomo- and hetero-functional, can be employed as a linker group. Couplingcan be affected, for example, through amino groups, carboxyl groups,sulfhydryl groups or oxidized carbohydrate residues.

In some embodiments, for example where an antimitotic agent is morepotent when free from the plant virus particle of the present invention,it can be desirable to use a linker group which is cleavable during orupon internalization into a cell, or which is gradually cleavable overtime in the extracellular environment. A number of different cleavablelinker groups have been described. The mechanisms for the intracellularrelease of a cytotoxic moiety agent from these linker groups includecleavage by reduction of a disulfide bond (e.g., U.S. Pat. No.4,489,710); by irradiation of a photolabile bond (e.g., U.S. Pat. No.4,625,014); by hydrolysis of derivatized amino acid side chains (e.g.,U.S. Pat. No. 4,638,045); by serum complement-mediated hydrolysis (e.g.,U.S. Pat. No. 4,671,958); and acid-catalyzed hydrolysis (e.g., U.S. Pat.No. 4,569,789).

It can be desirable to couple more than one type of antimitotic agentwithin a filamentous or rod-shaped plant virus particle of theinvention. By poly-derivatizing the plant viral particle of theinvention, several cytotoxic strategies can be simultaneouslyimplemented. For example, more than one type of anti-mitotic agent canbe coupled to a filamentous or rod-shaped plant virus particle.

Lymphoma Treatment

In one aspect, the present invention provides a method of treatinglymphoma in a subject by administering to the subject a therapeuticallyeffective amount of an anti-lymphoma virus particle. The anti-lymphomavirus particle can have any of the features described herein. Forexample, the plant virus particles can be tobacco mosaic virusparticles, and in some embodiments MMAE-based antimitotic agents can beused.

Filamentous or rod-shaped plant virus particles including antimitoticagents can be used to treat lymphoma, which is a type of cancer.“Cancer” refers to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (i.e., metastasize) as well as any of anumber of characteristic structural and/or molecular features. A “cancercell” refers to a cell undergoing early, intermediate or advanced stagesof multi-step neoplastic progression. The features of early,intermediate and advanced stages of neoplastic progression have beendescribed using microscopy. Cancer cells at each of the three stages ofneoplastic progression generally have abnormal karyotypes, includingtranslocations, inversion, deletions, isochromosomes, monosomies, andextra chromosomes. Cancer cells include “hyperplastic cells,” that is,cells in the early stages of malignant progression, “dysplastic cells,”that is, cells in the intermediate stages of neoplastic progression, and“neoplastic cells,” that is, cells in the advanced stages of neoplasticprogression.

Lymphoma is a group of blood cell tumors that develop from lymphaticcells. The two main categories of lymphomas are Hodgkin lymphomas andthe non-Hodgkin lymphomas (NHL). About 90% of lymphomas are non-Hodgkinlymphomas. Examples of non-Hodkin's lymphoma include diffuse largeB-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantlecell lymphoma, marginal zone B-cell lymphoma, burkitt lymphoma,lymphoplasmacytic lymphoma, hairy cell leukemia, primary central nervoussystem lymphoma, precursor T-lymphoblastic lymphoma and peripheralT-cell lymphomas. The most common type of lymphoma is B-cell lymphoma.Examples of non-Hodgkin's B-cell lymphoma include diffuse large B-celllymphoma, follicular lymphoma, marginal zone B-cell lymphoma ormucosa-associated lymphatic tissue lymphoma, small lymphocytic lymphoma(also known as chronic lymphocytic leukemia), and mantle cell lymphoma(MCL). Additional, more rare forms of B-cell lymphoma include Burkitt'slymphoma, lymphoplasmacytic lymphoma, nodal marginal zone B celllymphoma, splenic marginal zone lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis,primary central nervous system lymphoma, ALK-positive large B-celllymphoma, plasmablastic lymphoma, and large B-cell lymphoma arising inHHV8-associated multicentric Castleman's disease.

The filamentous or rod-shaped plant virus is used to target lymphoma ina subject. As used herein, targeting cancer tissue includes the abilityof the anti-lymphoma virus particles to reach and preferably accumulatein lymphoma after being administered to the subject. The ability offilamentous or rod-shaped plant virus particles to target cancer tissueis supported by the biodistribution studies carried out by theinventors. See International Patent Publication WO/2013/181557, thedisclosure of which is incorporated herein by reference. While notintending to be bound by theory, it currently appears that filamentousand rod-shaped plant virus particles are drawn to the leaky vasculaturecaused by the angiogenesis associated with rapid tumor growth, and thisleaky vasculature encourages entry for nanoparticles through smallpores, thereby delivering the anti-lymphoma plant virus particles to thelymphoma cells. As a result of this preferential accumulation,embodiments of the invention can deliver about 10%, about 20%, about30%, about 40%, or even about 50% or more of the injected dose to tumortissue.

In some embodiments, the subject being administered a therapeuticallyeffective amount of an anti-lymphoma plant virus particle is a subjectwho has been identified as having lymphoma. As is known to those skilledin the art, there are a variety of methods of identifying (i.e.,diagnosing) a subject who has lymphoma. Symptoms of lymphoma includelymphadenopathy or swelling of lymph nodes, which is the primarypresentation in lymphoma. Additional, systemic symptoms associated withboth Hodgkin lymphoma and non-Hodgkin lymphoma include fever, nightsweats, and weight loss. Lymphoma is definitively diagnosed by a lymphnode biopsy, meaning a partial or total excision of a lymph nodeexamined under the microscope, which reveals histopathological featuresthat may indicate lymphoma. After lymphoma is diagnosed, a variety oftests may be carried out to look for specific features characteristic ofdifferent types of lymphoma. These tests include: immunophenotyping,flow cytometry, and fluorescence in situ hybridization testing.

In some embodiments, the method further includes the step of ablatingthe lymphoma. Ablating the lymphoma can be accomplished using a methodselected from the group consisting of cryoablation, thermal ablation,radiotherapy, chemotherapy, radiofrequency ablation, electroporation,alcohol ablation, high intensity focused ultrasound, photodynamictherapy, administration of monoclonal antibodies, and administration ofimmunotoxins.

In some embodiments, the step ablating the lymphoma includesadministering a therapeutically effective amount of an anticancer agentcommonly used to treat lymphoma to the subject. Examples of anticanceragents commonly used to treat lymphoma include alkylating agents such ascyclophosphamide (Cytoxan®), chlorambucil, bendamustine (Treanda®), andifosfamide (Ifex®); Corticosteroids, such as prednisone anddexamethasone (Decadron®); platinum-based drugs such as cisplatin,carboplatin, and oxaliplatin; purine analogs such as fludarabine(Fludara®), pentostatin (Nipent®), and cladribine (2-CdA, Leustatin®);anti-metabolites such as cytarabine (ara-C), gemcitabine (Gemzar®),methotrexate, and pralatrexate (Folotyn®); and various other compoundssuch as vincristine (Oncovin®), doxorubicin (Adriamycin®), mitoxantrone,etoposide (VP-16), and bleomycin.

Targeting Moieties

In some embodiments, a targeting moiety can also be attached to thefilamentous or rod-shaped plant virus particle. By “targeting moiety”herein is meant a functional group which serves to target or direct thevirus particle to a particular location, cell type, diseased tissue, orassociation. In general, the targeting moiety is directed against atarget molecule. Thus, for example, antibodies, cell surface receptorligands and hormones, lipids, sugars and dextrans, alcohols, bile acids,fatty acids, amino acids, peptides and nucleic acids may all be attachedto localize or target the anti-lymphoma plant virus particle to aparticular site. In some embodiments, the targeting moiety allowstargeting of the plant virus particles of the invention to a particulartissue or the surface of a cell. Preferably, the targeting moiety islinked to the exterior surface of the virus to provide easier access tothe target molecule.

In some embodiments, the targeting moiety is a peptide. In furtherembodiments, the targeting moiety is an antibody. The term “antibody”includes antibody fragments, as are known in the art, including FabFab₂, single chain antibodies (Fv for example), chimeric antibodies,etc., either produced by the modification of whole antibodies or thosesynthesized de novo using recombinant DNA technologies. In furtherembodiments, the antibody targeting moieties of the invention arehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin.

In some embodiments, the antibody is directed against a cell-surfacemarker on a cancer cell; that is, the target molecule is a cell surfacemolecule. As is known in the art, there are a wide variety of cellsurface molecules known to be differentially expressed on tumor cells,including, but not limited to, HER2. Examples of physiologicallyrelevant carbohydrates may be used as cell-surface markers include, butare not limited to, antibodies against markers for breast cancer (CA15-3, CA 549, CA 27.29), mucin-like carcinoma associated antigen (MCA),ovarian cancer (CA125), pancreatic cancer (DE-PAN-2), and colorectal andpancreatic cancer (CA 19, CA 50, CA242). In some embodiments, a cellsurface molecule known to be differentially expressed on lymphoma cellsis used. Examples of such cell surface markers include CD20, CD22, andCD40.

Coatings on the Virus Particle Exterior

In some embodiments, a coating can be added to the exterior of the plantvirus particle to improve bioavailability. Administering plant virusparticles to a subject can sometimes generate an immune response. An“immune response” refers to the concerted action of lymphocytes, antigenpresenting cells, phagocytic cells, granulocytes, and solublemacromolecules produced by the above cells or the liver (includingantibodies, cytokines, and complement) that results in selective damageto, destruction of, or elimination from the human body of cancerouscells, metastatic tumor cells, invading pathogens, cells or tissuesinfected with pathogens, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues. Components of an immuneresponse can be detected in vitro by various methods that are well knownto those of ordinary skill in the art.

Generation of an immune response by the anti-lymphoma virus particles istypically undesirable. Accordingly, in some embodiments it may bepreferable to modify the exterior of the plant virus particle or takeother steps to decrease the immune response. For example, animmunosuppressant compound can be administered to decrease the immuneresponse. More preferably, the anti-lymphoma virus particle can bemodified to decrease its immunogenicity. Examples of methods suitablefor decreasing immunity include attachment of anti-fouling (e.g.,zwitterionic) polymers, glycosylation of the virus carrier, andPEGylation.

In some embodiments, the immunogenicity of the anti-lymphoma virusparticle is decreased by PEGylation. PEGylation is the process ofcovalent attachment of polyethylene glycol (PEG) polymer chains to amolecule such as a filamentous plant virus carrier. PEGylation can beachieved by incubation of a reactive derivative of PEG with the plantvirus particle exterior. The covalent attachment of PEG to theanti-lymphoma virus particle can “mask” the agent from the host's immunesystem, and reduce production of antibodies against the carrier.PEGylation also may provide other benefits. PEGylation can be used tovary the circulation time of the filamentous plant virus carrier. Forexample, use of PEG 5,000 can provide an anti-lymphoma virus particlewith a circulation half-life of about 12.5 minutes, while use of PEG20,000 can provide an anti-lymphoma virus particle with a circulationhalf life of about 110 minutes.

The first step of PEGylation is providing suitable functionalization ofthe PEG polymer at one or both terminal positions of the polymer. Thechemically active or activated derivatives of the PEG polymer areprepared to attach the PEG to the anti-lymphoma virus particle. Thereare generally two methods that can be used to carry out PEGylation; asolution phase batch process and an on-column fed-batch process. Thesimple and commonly adopted batch process involves the mixing ofreagents together in a suitable buffer solution, preferably at atemperature between 4 and 6° C., followed by the separation andpurification of the desired product using a chromatographic technique.

Administration and Formulation of Anti-lymphoma Plant Virus Particles

In some embodiments, the anti-lymphoma virus particle is administeredtogether with a pharmaceutically acceptable carrier to provide apharmaceutical formulation. Pharmaceutically acceptable carriers enablethe anti-lymphoma virus particle to be delivered to the subject in aneffective manner while minimizing side effects, and can include avariety of diluents or excipients known to those of ordinary skill inthe art. Formulations include, but are not limited to, those suitablefor oral, rectal, vaginal, topical, nasal, ophthalmic, or parental(including subcutaneous, intramuscular, intraperitoneal, intratumoral,and intravenous) administration. For example, for parenteraladministration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit activity of the compound, can be used. Other suitablecarriers include, but are not limited to, alcohol, phosphate bufferedsaline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the anti-lymphomavirus particle into association with a pharmaceutically acceptablecarrier that constitutes one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theanti-lymphoma virus particle into association with a liquid carrier, afinely divided solid carrier, or both, and then, if necessary, shapingthe product into the desired formulations. The methods of the inventioninclude administering to a subject, preferably a mammal, and morepreferably a human, the composition of the invention in an amounteffective to produce the desired effect. The formulated anti-lymphomavirus particle can be administered as a single dose or in multipledoses.

Useful dosages of the antimitotic agents and anti-lymphoma virusparticles can be determined by comparing their in vitro activity and thein vivo activity in animal models. Methods for extrapolation ofeffective dosages in mice, and other animals, to humans are known in theart; for example, see U.S. Pat. No. 4,938,949. An amount adequate toaccomplish therapeutic or prophylactic treatment is defined as atherapeutically- or prophylactically-effective dose. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until an effect has been achieved. Effective doses ofthe anti-lymphoma virus particles vary depending upon many differentfactors, including means of administration, target site, physiologicalstate of the patient, whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic.

For administration of the anti-lymphoma virus particles for treatment oflymphoma in a subject, the dosage of the antic-mitotic agent ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg. A suitable amount ofanti-lymphoma virus particle is used to provide the desired dosage. Anexemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months. The anti-lymphomavirus particle is usually administered on multiple occasions.Alternatively, the anti-lymphoma virus particle can be administered as asustained release formulation, in which case less frequentadministration is required. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patent can be administered a prophylacticregime.

The compositions can also include, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes).

For parenteral administration, compositions of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.

The present invention is illustrated by the following example. It is tobe understood that the particular example, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLE Example 1: Delivery of Potent Anti-Mitotic ChemotherapeuticUsing High Aspect Ratio, Soft Matter Nanoparticles

A study of the development and testing of an anti-mitotic soft mattervirus-based nanoparticle (VNP) using the nucleoprotein components of thetobacco mosaic virus (TMV, FIG. 1) is described. TMV is a plant virusthat has several properties that it a favorable nanoparticle choice forbiomedical applications. TMV offers a stable, monodisperse, andbiocompatible protein-based scaffold. J. K. Pokorski and N. F.Steinmetz, Mol. Pharm., vol. 8, no. 1, pp. 29-43 (2011); Wen et al., J.Vis. Exp. JoVE, no. 69 (2012). The TMV structure is known to an atomicresolution, this allows the biomedical engineer and chemist to identifyspecific reactive groups to be targeted for bioconjugation of medicalcargo such as drug or contrast agents and display of targeting ligandsor shielding molecules. To be specific, TMV is a 300 nm by 18 nm rigidhollow nanotube with a 4 nm-wide interior channel (see FIG. 1). The rodshaped, soft matter nanoparticle consists of 2130 identical coatproteins. The reactive groups on the exterior or interior channel areknown: with tyrosine 139 on the exterior, and glutamine 97 and glutamine106 on the interior. Furthermore, genetic engineering, allows theintroduction of new functionality. One example of this is thelysine-added mutant ‘TMV-lysine’ that displays reactive lysine groups onthe exterior protein shell, and is easily modifiable using well knownN-Hydroxysuccinimide (NHS) chemistry (TMV-lys, FIG. 1). To conclude, TMVoffers a modifiable, uniform, and organic nanoparticle scaffold to builda therapeutic nanoformulation.

In a previous study, the inventors observed that TMV was taken up by thecervical cancer cell line HeLa; uptake was by endocytosis targeting theTMV-based nanoparticle into the endolysosomal compartment. In this lowpH environment with highly reactive protease and hydrolase, theparticles were left intact over the time course studied (1 week), butthe chemically bio-conjugated molecules on the exterior and interiorwere rapidly cleaved. Wen et al., Bioconjug. Chem., vol. 26, no. 1, pp.51-62 (2015). Based on these previous findings, the inventorshypothesized that they could make use of the natural cancer cell uptakeand fate of the TMV carrier to deliver an anti-mitotic drug cargo thatcould be activated and released within the cellular endolysosome.Therefore, when in the conjugated form the drug would be renderednon-active and therefore safe—and would only induce toxicity upon cancercell targeting and activation.

The anti-mitotic drug: vcMMAE, valine-citrulline monomethyl auristatin E(see FIG. 2) was selected. vcMMAE consists of a lysosomally cleavabledipeptide, valine-citrulline, and the anti-mitotic agent, MMAE. Oncecleaved, the MMAE drug is cytotoxic through inhibiting thepolymerization of tubulin, which stops the cell from replicating.

Thus far VNP technologies have primarily focused on the delivery ofdrugs targeting solid tumors. As a new direction, the inventorsdeveloped soft matter nanoparticles for applications targeting lymphomaand delivering vcMMAE.

Methods

Propagation and Purification of TMV-lysine. TMV-lysine (TMV-lys) is agenetically modified TMV where reactive lysine groups were introduced onthe ends of the coat proteins. Smith et al., Virology, vol. 348, no. 2,pp. 475-488 (2006). TMV-lys was propagated using Nicotiana benthamianaplants (a tobacco plant species). The virus was isolated via establishedpurification procedures yielding up to 100 milligrams of TMV-lys per 100grams of infected tobacco plant leaves. Verch et al., J. Immunol.Methods, vol. 220, no. 1-2, pp. 69-75 (1998).

UV/VIS Spectroscopy. A Thermo Scientific NanoDrop 2000 Spectrophotometerwas used to measure the concentration of TMV using the TMV specificextinction coefficient (1.36 M⁻¹cm⁻¹), the known path length (0.1 cm⁻¹)and the Beer-Lambert equation. The Beer-Lambert law is defined asA=ε*d*c, where ε is the extinction coefficient, A is the absorbancemeasured at a defined wavelength, d is the path length, and c is theconcentration. To solve for concentration we rearrange the equation asA/(ε*d).

Chemical Conjugation. TMV-lys was reacted with 10 molar excess ofN-succinmidyl-S-acetylthiopropionate (SATP) (Thermo Fisher) with 10%(v/v) dimethyl sulfide (DMSO) in 10 mM potassium phosphate buffer (pH7.0). The resulting TMV-SATP complex was purified via ultracentrifuge,reacted with 10% deacetylation solution (v/v), (5 M Hydroxylamine, 25 mMEDTA in PBS, pH 7.4), for two hours, and then purified with a PD-10desalting column (GE healthcare life sciences). Next, the TMV-SATPsolution was reacted with 10 molar excess of vcMMAE (Med Chem Express)overnight and purified via ultracentrifuge. Yields after ultracentrifugepurification range from 70%-90%, and yields after desalting column rangefrom 30%-50%.

SDS-PAGE. Coat proteins were analyzed using 4-12% NuPAGE gels(Invitrogen) using 1x (Nmorpholino) propanesulfonic acid (MOPS) runningbuffer (Invitrogen). 20 μg of protein with 4X loading LDS dye was added,and denatured through heating at 100° C. for 5 minutes. Afterseparation, the gel was first placing the gel in destain solution (10%acetic acid, 50% methanol, and 40% H₂O) for 30 minutes. 10 mL of theused destain solution was then diluted with 40 mL H₂O and 50 μL ofCoomassie Blue R250. The gel was placed in this solution for 30 minutesto stain for protein, then immediately photographed using an AlphaImager(Biosciences) imaging system.

Transmission electron microscopy(TEM). 2 μL drops of 0.1 mg/mLTMV-vcMMAE in H₂O was placed onto TEM grids and allowed to dry. The gridwas then washed in DI water, and stained with 2% (w/v) uranyl acetatefor two minutes. After drying, samples were examined using a Zeiss Libra200FE transmission electron microscope operated at 200 kV.

Cell culture. Karpas 299 cells (ATCC) were maintained in RPMI-1640 at37° C. in a 5% CO2 humidified atmosphere. The medium was supplementedwith 20% (v/v) heat-inactivated fetal bovine serum (FBS), and 1% (v/v)penicillin-streptomycin. All reagents were obtained from Gibco.

Cell viability assay. Cells at a concentration of 10,000 cells/mL wereseeded in a sterile, tissue culture-treated, 96-well clear bottom platefor 24 hours at 37° C. in a 5% CO₂ humidified atmosphere. Cells werethen incubated with fresh media with 0.02 nM to 200 nM VcMMAE for 72hours. At 92 hours, Alamar Blue (ThermoFisher) was added, and cellviability was measured at 96 hours according to the manufacturer'sinstructions using a fluorescence plate reader with emission at 540 nMand excitation at 610 nM.

Confocal Microscopy. Karpas 299 cells were seeded at a density of500,000 cells/well in a 96 well plate in fresh RPMI and incubated withTMV at 1,000,000 particles/cells for 8 hours at 37° C. in a 5% CO₂humidified atmosphere. Cells were spun down at 500 g and washed twotimes with CELL buffer (0.1 mL 0.5 M EDTA, 0.5 mL fetal bovine serum,1.25 mL 1 M HEPES pH 7.0, 48.15 mL PBS), and then fixed using 2% (v/v)paraformaldehyde in CELL buffer, then washed an additional two times inCELL buffer. Cells were washed in PB buffer (0.2% (v/v) Triton X-100 inDPBS) twice, and incubated with rabbit anti-TMV (1:500) (PacificImmunology) and mouse anti-human LAMP-1 (1:500) (Sigma Aldrich) in CELLbuffer for an hour. The cells were then washed two more times with PBbuffer and spun down on cover slips at 2,000 rpm for 5 minutes. Then thecells were incubated with secondary antibodies using AlexaFluor488-labeled goat anti-mouse antibody and AlexaFluor 555 anti-rabbitantibody (Invitrogen) in PB with 5% (v/v) goat serum for one hour.Lastly, the slips were washed two more times with CELL buffer, and thenstained with DAPI (Sigma Aldrich) and imaged at 40X on an OlympusFluo-View™ FV1000 LSCM microscope.

Results and Discussion

TMV-lys was first propagated in N benthamiana plants. TMV-lys is alysine-added mutant of TMV which displays a lysine residue instead of aserine at amino acid position 158; the amine-functional lysine group istherefore solvent-exposed and located at the C-terminus of the coatprotein (see FIG. 1). Geiger et al., Nanoscale, vol. 5, no. 9, pp.3808-3816 (2013) TMV-lys was extracted in yields of 1 mg of pure virusper gram of infected leaves using established virus purificationtechniques.

To obtain vcMMAE-loaded TMV, the anti-mitotic drug was bioconjugatedusing a three-step reaction (FIG. 2A). First, the amine handle of theTMV-lys was converted into a acetate-protected sulfhydryl group usingthe bi-functional linker N-succinimidyl-S-acetylthiopropionate (SATP),using a ratio of SATP:TMV of 15:1 (FIG. 2A). The reaction was purifiedvia ultracentrifugation over a 40% (w-v) sucrose cushion. The TMV-SATPending sulfhydryl group was then deprotected using hydroxylamine andpurified using a PD-10 desalting column, yielding a free thiol handlefor conjugation with the maleimide-terminated vcMMAE throughthiol-Michael reactions; using a ratio of vcMMAE:TMV of 10:1. Thestructure of vcMMAE is shown in FIG. 2B. The reaction mix again waspurified by ultracentrifugation over sucrose cushion and the finalformulation was characterized by UV-VIS, SDS-gel, and TEM (FIGS. 3A-C).

UV/VIS spectroscopy was conducted to analyze the amount of proteinrecovered. Reactions starting with 5 mg of TMV-lys resulted in yields ofapproximately 3.7 mg of TMV-SATP, and yields of 1.3 mg of TMV-vcMMAE.This means that 75% was recovered in step 1 and only 35% of particleswere recovered in step 2.

The particles were characterized after each conjugation step usingSDS-PAGE (FIG. 3A). Using this technique, the TMV particle is denaturedand disassembled into its coat proteins, which are analyzed byelectrophoresis through a polyacrylamide matrix. Using this method, thecoat proteins are separated by electrophoretic mobility based onmolecular weight. Differences in the bands pattern indicate differencesin the molecular weight of the various compositions of coat protein (CP)vs. CP-SATP vs. CP-vcMMAE. The band pattern reveals the molecular weightof the CPs and therefore indicates whether the drug cargo is attached.

The TMV coat protein measures 17.5-kDa and is detectable on the SDS-PAGE(Lane 1, FIG. 3A). A small percentage of coat protein dimers are alsodetectable at ˜40-kDa molecular weight standard. The dimers are aresults of inter-coat protein crosslinking based on disfulfide bridges.Shifts in the band pattern indicate successful conjugation of SATP andthe drug vcMMAE (FIG. 3B). The SATP linker has a molecular weight of245.25 g/mol; a negligible shift toward higher molecular weight bandsindicates successful conjugation, however, it should be noted that theSDS PAGE methods does not provide the resolution to resolve the smalldifference in the molecular weight change of 0.5 kDa comparing the CPvs. CP-STAP. The drug, vcMMAE, has a molecular weight of 1316.64 g/mol;a shift towards the higher molecular weight band indicates thatconjugation was successful, and the CP-vcMMAE band is detectable at 20kDa.

The SDS-gel displayed distinct bands comparing lane 1, TMV, and lane 5,TMVvcMMAE, indicating that a higher molecular weight complex waspresent. Densinometric analysis conducted using ImageJ showed ˜100% bandseparation. The uniform shift of the band indicates complete conversionof the amines to thiols, followed by drug loading, i.e. the SDS-PAGEindicates that each of the 2,130 available sites were labeled with thetherapeutic cargo (FIG. 3B).

Finally, transmission electron microscopy (TEM) revealed that theTMVvcMMAE formulations remained intact; imaging of negatively-stainedTEM grids showed structurally sound TMV nanoparticles with dimensions of300×18 nm (FIG. 3C). TEM images showed that the TMV rods were intact andnon-aggregated after the bioconjugation and purification processes.

To conclude discussion of the synthesis of the vcMMAE-conjugated TMV;the inventors have developed a protocol allowing efficient drug loadingof TMV. The chemical reaction reached completion with each of the 2,130coat proteins being modified with a drug molecule. On a %-molecularweight basis, this translates to a drug loading efficiency of ˜7% wt perTMV. This is comparable to immunotherapies which have drug loading of upto ˜7% wt per IgG, and up to ˜10% wt per diabody in antibody conjugatedMMAE studies. Rink et al., Curr. Opin. Oncol., vol. 25, no. 6, pp.646-651 (2013). Nevertheless, antibody-drug conjugates are limited toonly carrying up to 8 drug molecules per antibody, which is far fewerthan the load which TMV can carry, namely ˜2,000 copies of the payload.

The inventors next set out to study the in vitro properties of the drugcandidate using Karpas 299 cells, a human derived non-Hodgkin's large Bcell lymphoma, and the fate of the TMV formulation in these cells, andsecond, drug efficacy.

A cell uptake and colocalization study was carried out to evaluatewhether TMV would target and be taken up by Karpas 299 cells, andwhether the particles would be targeted to the endolysosomalcompartment. First, Karpas 299 cells were incubated with TMV for 8 hoursusing one million TMV particles per cell. Then the cells were washed toremove excess TMV, followed by staining of TMV using TMV-specificantibodies and secondary dye-labeled antibodies. At the same time, theendolyosome was stained using LAMP-1 staining. The cells were thenfixed, mounted and imaged by fluorescence microscopy (FIG. 4).

As seen in FIG. 4, the red fluorescent antibodies tagging TMV overlapwith the green antibodies tagging the endolysosome. Thus the confocalmicroscopy imaging confirmed that the Karpas 299 cells uptake TMV, andthat the nanoparticles traffic to the endolysosome. Based on thesefindings, I hypothesize that the TMV-vcMMAE would also be taken up bythe Karpas 299 cells targeting the endolysosome. The vcMMAE drug isdesigned to be activated and cleaved within the endolysosomalcompartment. Specifically the valine-citruline (vc)-linker is aprotease-specific cleavable linker, resulting in release of the activeMMAE component.

To evaluate the efficacy of TMV-vcMMAE, cell viability assays wereperformed using Alamar Blue assay Alamar Blue reagent is primarily madeof resazurin, a nontoxic cell permeable non-fluorescent blue molecule.Upon entering viable cells, resazurin is reduced to resorufin, which isa bright red fluorescent molecule. Protocols for using the reagent areavailable online.

Karpas 299 cells were seeded for 24 hours and then incubated with freevcMMAE or TMV-MMAE for 72 hours. At 92 hours, Alamar Blue was added andthe cells fluorescence was measured after 4 hours of incubation at 540nm excitation and 610 nm emission. The results for both free vcMMAE andTMV-MMAE are plotted in FIG. 4, and the ICs₅₀ values were calculated.The IC₅₀ value defines the drug concentration where half of the cellsare killed. The IC50 for free vcMMAE was determined to be about 25.8 nM,and the ICs₅₀ for TMV-MMAE was found to be 256.1 nM (FIG. 5).

IC₅₀ values of the free vcMMAE drug vary in the literature depending onthe set up of the assay, e.g. time course and cell lines used, and arefound in the low nanomolar range at around 5 nM. Wen et al., Bioconjug.Chem., vol. 26, no. 1, pp. 51-62 (2015). IC₅₀ values of antibody drugconjugates are generally found ranging from 2 nM to 42 nM. The inventorssaw a similar trend, with a low IC₅₀ of the free drug, and an order ofmagnitude higher IC₅₀ using VNP loaded with drug. The lower IC₅₀ valuesof the nanoparticle conjugates may be explained by lower cell uptakekinetics, as has been reported with other systems. Danhier et al., J.Controlled Release, vol. 133, no. 1, pp. 11-17 (2009); Panyam and V.Labhasetwar, Adv. Drug Deliv. Rev., vol. 55, no. 3, pp. 329-347 (2003).Nevertheless, the inventors expect that the nanoparticle-assistedtargeted drug delivery approach will outperform the in vivo profiles ofthe free drug through enhanced biodistribution and pharmacokinetics.

CONCLUSIONS

The inventors demonstrated that TMV could be loaded with a high payloadof vcMMAE, with all of its coat proteins labeled with drug for treatmentof lymphoma. The particles are taken up by cancer cells and are targetedto the endolysosomal compartment, where the drug is then cleaved fromthe VNP. It was found that TMV-delivered vcMMAE was effective in killingcancer cells in vitro and demonstrated an IC₅₀ in the nanomolar range.The inventors found that the TMV-vcMMAE complex had a higher IC₅₀ valuethan free vcMMAE. One explanation why the efficacy of the viralnanoparticle may be lower is due to lower cell uptake kinetics.

One of the next steps for the optimization of the TMVvcMMAE therapeuticwould be to give the particles further selectivity. This can be achievedthrough conjugation of targeting peptides or antibodies. For example,direction the drug-loaded nanoparticles with specificity for markers ofdiffuse large B cell lymphoma is expected to increase cell uptake ratesand selectivity. CD20, CD22, and CD40 have been identified as candidatetargets and nanoparticle formulations with selectivity for these targetsare under development for treatment of B-cell lymphomas. Rillahan etal., Chem. Sci., vol. 5, no. 6, pp. 2398-2406 (2014); Holder et al.,Eur. J. Immunol., vol. 23, no. 9, pp. 2368-2371 (1993); T. M. Allen,Nat. Rev. Cancer, vol. 2, no. 10, pp. 750-763 (2002).

Antibodies with specificity toward these markers are also indevelopment, with some formulations already being used in the clinic fortreatment of lymphoma. However, antibodies are limited in only beingable to carry up to 8 drug molecules. TMV provides advantages, becausethis nanoparticle formulation can carry more than 2,000 drugs on itsexterior. Furthermore, the interior channel can also be used forconjugation of drugs or contrast agents—toward the development oftheranostics. This high number of reactive sites opens up greaterflexibility compared to antibody drug conjugate therapies. Bio27conjugation of antibodies to the TMV-vcMMAE formulation is expected tobe a powerful tool for targeted drug delivery targeting Non-Hodgkin'slymphoma. In conclusion, there are many promising avenues for thefurther development of virus-based nanoparticle therapeutics targetingsoft-cancer applications.

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. In particular,the inventors are not bound by theories described herein. The inventionis not limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

What is claimed is:
 1. An anti-lymphoma virus particle, comprising afilamentous or rod-shaped plant virus particle linked to an antimitoticagent.
 2. The anti-lymphoma virus particle of claim 1, wherein the plantvirus particle is a filamentous plant virus particle.
 3. Theanti-lymphoma virus particle of claim 2, wherein the filamentous plantvirus particle the Alphaflexiviridae family
 4. The anti-lymphoma virusparticle of claim 1, wherein the plant virus particle is a rod-shapedplant virus particle.
 5. The anti-lymphoma virus particle of claim 4,wherein the plant virus particle is a member of the Virgaviridae family.6. The anti-lymphoma virus particle of claim 4, wherein the plant virusparticle is a tobacco mosaic virus.
 7. The anti-lymphoma virus particleof claim 1, wherein the exterior surface of the plant virus particle hasbeen PEGylated.
 8. The anti-lymphoma virus particle of claim 1, whereinthe antimitotic agent is a dolastatin.
 9. The anti-lymphoma virusparticle of claim 8, wherein the dolastatin is monomethyl auristatin E(MMAE).
 10. The anti-lymphoma virus particle of claim 8, wherein thedolastatin is valine-citrulline monomethyl auristatin E (vcMMAE). 11.The anti-lymphoma virus particle of claim 1, wherein the antimitoticagent is covalently conjugated to the exterior of the plant virusparticle.
 12. The anti-lymphoma virus particle of claim 1, wherein atargeting ligand is attached to the exterior of the plant virusparticle.
 13. A method of treating lymphoma in a subject byadministering to the subject a therapeutically effective amount of ananti-lymphoma virus particle, comprising a filamentous or rod-shapedplant virus particle linked to an antimitotic agent.
 14. The method ofclaim 13, wherein the lymphoma is non-Hodgkin's lymphoma.
 15. The methodof claim 13, wherein the anti-lymphoma virus particle is administeredtogether with a pharmaceutically acceptable carrier.
 16. The method ofclaim 13, wherein the plant virus particle is a rod-shaped plant virusparticle.
 17. The method of claim 13, wherein the antimitotic agent is adolastatin.
 18. The method of claim 17, wherein the doloastatin is MMAE.19. The method of claim 17, wherein the dolastatin is vcMMAE.
 20. Themethod of claim 13, wherein the antimitotic agent is covalentlyconjugated to the exterior of the plant virus particle.
 21. The methodof claim 13, wherein a targeting ligand is attached to the exterior ofthe plant virus particle.